This is a renewal application for the University of Maryland Baltimore Biomarker Reference Laboratory (UMB BRL). Over the past 5 years the UMB BRL (PI, Sanford Stass, MD) have been active collaborating and supporting the Early Detection Research Network (EDRN). The PI participated on multiple EDRN committees/activities. The UMB BRL has provided resources for analytical and clinical validation of biomarkers, assay development/technologies, and standardization of assay methods in collaboration with multiple investigators. The UMB BRL is organized as a team of directors/co-investigators with broad EDRN knowledge, experience, expertise and provides a wide range of core pathology laboratories with CLIA and/or CAP accreditation including next generation sequencing, clinical chemistry, molecular diagnostics, flow cytometry, cytogenetics, and GLP compliant laboratories. There are also core laboratories including genomics, mass spectrometry, NMR tissue bank, and biostatistics. There is a product development study, with the objective to develop targeted next generation sequencing (NGS) using Ion Torrent (ITO) for miRNAs biomarkers (developed here) in sputum obtained with the Lung Flute (LF) for the early detection of non-small-cell lung cancer in patients with nodules on CT scan. There are 3 specific aims: (1) To validate whether the 102 miRNA biomarker candidates of lung cancer we recently identified, which include 2 miRNAs from our previous EDRN study, can be analyzed in lung tumor samples by using the IoT-targeted NGS platform in a CLIA accredited laboratory. (2) To optimize a panel of highly specific and sensitive sputum miRNA biomarkers for identifying malignant pulmonary nodules using our retrospective sputum specimens with the IoT platform. (3) To validate analytical and clinical performance of the biomarker panel using the IoT in prospectively collected sputum samples from the University of Maryland, Vanderbilt-Ingram Cancer Center, and the New York University Langone Medical Center using the Lung Flute. The UMB BRL leverages the experience of its multi-disciplinary investigators and considerable expertise of the leadership to provide flexible support to the EDRN for standardized procedures, high throughput, robust assays, clinical translation, design and conduct analytical validation studies, a variety of laboratory/platform assays, GLP compliance, and adherence to regulatory requirements including CLIA/CAP, FDA, and GLP. Numerous collaborations are within and outside UMB guaranteeing maximum responsiveness to the EDRN. Lines of authority/organizational structure in the UMB BRL ensure timely performance, efficient communication and rapid translation of findings from the development phase into clinical application.